Development of microsatellite markers for sister species Linum suffruticosum and Linum tenuifolium in their overlapping ranges.

BACKGROUND: Microsatellite markers were developed for distylous Linum suffruticosum and tested in the monomorphic sister species Linum tenuifolium. These species are perennial herbs endemic to the western and northwestern Mediterranean, respectively, with a partially overlapping distribution area. METHODS AND RESULTS: We developed 12 microsatellite markers for L. suffruticosum using next generation sequencing, and assessed their polymorphism and genetic diversity in 152 individuals from seven natural populations. The markers displayed high polymorphism, with two to 16 alleles per locus and population, and average observed and expected heterozygosities of 0.833 and 0.692, respectively. All loci amplified successfully in the sister species L. tenuifolium, and 150 individuals from seven populations were also screened. The polymorphism exhibited was high, with two to ten alleles per locus and population, and average observed and expected heterozygosities of 0.77 and 0.62, respectively. CONCLUSIONS: The microsatellite markers identified in L. suffruticosum and tested in L. tenuifolium are a powerful tool to facilitate future investigations of the population genetics, mating patterns and hybridization between both Linum species in their contact zone.

Asymmetries of reproductive isolation are reflected in directionalities of hybridization: integrative evidence on the complexity of species boundaries.

The complex nature of species boundaries has been a central topic in evolutionary biology ever since Darwin. Despite numerous separate studies on reproductive isolation and hybridization, their relationship remains underinvestigated. Are the strengths and asymmetries of reproductive barriers reflected in the extent and directionalities of interspecific genetic exchange? We combined field, experimental, and molecular data to quantify strengths and asymmetries of sympatric reproductive barriers and hybridization between florally heteromorphic primroses. We also assessed whether generalist pollinators discriminate between different floral cues and contribute to reproductive isolation, a long-debated topic. Sympatric reproductive isolation is high but incomplete, and most phenotypic intermediates are genetic F1 hybrids, whereas backcrosses are rare, revealing low interspecific gene flow. Species integrity rests on multiple barriers, but ethological isolation is among the strongest, demonstrating that even generalist pollinators crucially contribute to the maintenance of species boundaries. Furthermore, reproductive barriers are weaker for Primula veris and short-styled plants, results corroborated by molecular data. Thus, in florally heteromorphic systems, both species- and morph-dependent asymmetries affect permeability of species boundaries. Our study illustrates how the interactions between complex floral syndromes and pollinators shape species boundaries in unique, previously undescribed ways.

Pollination, mating and reproductive fitness in a plant population with bimodal floral-tube length.

Mating patterns and natural selection play important roles in determining whether genetic polymorphisms are maintained or lost. Here, we document an atypical population of Lapeirousia anceps (Iridaceae) with a bimodal distribution of floral-tube length and investigate the reproductive mechanisms associated with this pattern of variation. Flowers were visited exclusively by the long-proboscid fly Moegistorhynchus longirostris (Nemestrinidae), which exhibited a unimodal distribution of proboscis length and displayed a preference for long-tubed phenotypes. Despite being visited by a single pollinator species, allozyme markers revealed significant genetic differentiation between open-pollinated progeny of long- and short-tubed phenotypes suggesting mating barriers between them. We obtained direct evidence for mating barriers between the floral-tube phenotypes through observations of pollinator foraging, controlled hand pollinations and measurements of pollen competition and seed set. Intermediate tube-length phenotypes produced fewer seeds in the field than either long- or short-tubed phenotypes. Although floral-tube length bimodality may not be a stable state over long timescales, reproductive barriers to mating and low 'hybrid' fitness have the potential to contribute to the maintenance of this state in the short term.

Floral polymorphism and the fitness implications of attracting pollinating and florivorous insects.

BACKGROUND AND AIMS: Floral polymorphism is frequently attributed to pollinator-mediated selection. Multiple studies, however, have revealed the importance of non-pollinating visitors in floral evolution. Using the polymorphic annual daisy Ursinia calenduliflora, this study investigated the importance of different insect visitors, and their effects on fitness, in the maintenance of floral polymorphism. METHODS: The spatial structure of a discrete floral polymorphism was characterized based on the presence/absence of anthocyanin floret spots in U. calenduliflora. A 3-year observational study was then conducted in polymorphic populations to investigate differences in visitation rates of dominant visitors to floral morphs. Experiments were performed to explore the floral preference of male and female Megapalpus capensis (the dominant insect visitor) and their effectiveness as pollinators. Next, floral damage by antagonistic florivores and the reproductive success of the two floral morphs were surveyed in multiple populations and years. KEY RESULTS: Floral polymorphism in U. calenduliflora was structured spatially, as were insect visitation patterns. Megapalpus capensis males were the dominant visitors and exhibited strong preference for the spotted morph in natural and experimental observations. While this may indicate potential fitness benefits for the spotted morph, female fitness did not differ between floral morphs. However, as M. capensis males are very efficient at exporting U. calenduliflora pollen, their preference may likely increase the reproductive fitness of the spotted morph through male fitness components. The spotted morph, however, also suffered significantly greater costs due to ovule predation by florivores than the spotless morph. CONCLUSIONS: The results suggest that pollinators and florivores may potentially exert antagonistic selection that could contribute to the maintenance of floral polymorphism across the range of U. calenduliflora. The relative strength of selection imposed by each agent is potentially determined by insect community composition and abundance at each site, highlighting the importance of community context in the evolution of floral phenotypes.

Floral polymorphism in Chamaecrista flexuosa (Fabaceae-Caesalpinioideae): a possible case of atypical enantiostyly?

BACKGROUND AND AIMS: Reciprocal herkogamy, including enantiostyly and heterostyly, involves reciprocity in the relative positions of the sexual elements within the flower. Such systems result in morphologically and, since pollen is deposited on and captured from different parts of the pollinator, functionally distinct floral forms. Deviations from the basic pattern may modify the functionality of these mechanisms. For heterostylous species, such deviations are generally related to environmental disturbances, pollination services and/or reduced numbers of one floral morph. Deviations for enantiostylous species have not yet been reported. This study aims to investigate enantiostyly in Chamaecrista flexuosa, in particular the presence of deviations from the standard form, in an area of coastal vegetation in north-east Brazil. METHODS: Observations and investigations of floral biology, the reproductive system, pollinator behaviour, floral morphology and morphometry were performed. KEY RESULTS: In C. flexuosa flowers, anthers of different size but similar function are grouped. The flowers were self-compatible and set fruits after every treatment, except in the spontaneous self-pollination experiment, thereby indicating their dependence on pollen vectors. The flowers were pollinated by bees, especially Xylocopa cearensis and X. grisencens. Pollen is deposited and captured from the ventral portion of the pollinator's body. Variations in the spatial arrangement of floral elements allowed for the identification of floral morphs based on both morphological and functional criteria. Using morphological criteria, morphologically right (MR) and morphologically left (ML) floral morphs were identified. Three floral morphs were identified using functional criteria: functionally right (FR), functionally central (FC) and functionally left (FL). Combinations of morphologically and functionally defined morphs did not occur in equal proportions. There was a reduced frequency of the MR-FR combination. CONCLUSIONS: The results indicate the occurrence of an atypical enantiostyly in C. flexuosa. This seems to improve reproductive success by increasing the efficiency of pollen deposition and capture.

Variation in pollen sterility and gender specialization: an investigation with distylous species of Erythroxylum (Erythroxylaceae).

Differences in pollen sterility between morphs in distylous populations may represent the first step in the evolution of gender specialization. Theoretically, for partially or completely male sterile individuals to persist they must have some fitness advantage that compensates for the loss of male function. Gender specialization is considered a widespread process in Erythroxylum, but male sterility and the resource reallocation to female function have been investigated in few species and populations. In 18 populations of four distylous species of Erythroxylum, we quantified the levels of male sterility, estimated through pollen sterility, in short- and long-styled flowers to test if sterility is morph-biased. In one population per species, we also described the frequency of floral visitors, the production of flowers and fruits, and the quality of fruits and seeds of short- and long-styled plants to evaluate the expression of trade-offs in allocation to male and female function. In some populations of E. campestre and E. deciduum, short-styled flowers possessed higher levels of pollen sterility than long-styled flowers. Although most flowers of E. suberosum and E. tortuosum also expressed pollen sterility, the frequency of sterility was similar between morphs in all populations. Differences in reproductive output between morphs occurred only in populations of species with morph-biased sterility, but none of the variations reflect resource allocation to female fitness of short-styled plants. Differences in the level of sterility between morphs indicates the potential for gender specialization in populations of E. campestre and E. deciduum, despite the apparent lack of a trade-off in allocation.

Cell number explains the intraspecific spur-length variation in an Aquilegia species.

Variations of nectar spur length allow pollinators to utilize resources in novel ways, leading to the different selective pressures on spurs and allowing taxa to diversify. However, the mechanisms underlying spur length variation remain unclear. Interspecific comparisons of spur length suggest that both cell division and anisotropic expansion could explain the changes of spur length, and that hormone-related genes contribute to the process of spur formation. In contrast, little is known about intraspecific spur length variation. In Aquilegia rockii, spur length varies strikingly, ranging from 1 mm to 18 mm. To examine the potential mechanisms underlying spur length variation in A. rockii, we observed cell morphology and analyzed RNA-seq of short- and long-spurred flowers. Scanning electron microscopy revealed that at two positions on spurs there were no differences in either cell density or cell anisotropy between short- and long-spurred flowers, suggesting that in A. rockii changes in cell number may explain variations in spur length. In addition, we screened transcriptomes of short- and long-spurred flowers for differentially expressed genes; this screen identified several genes linked to cell division (e.g., F-box, CDKB2-2, and LST8), a finding which is consistent with our analysis of the cellular morphology of spurs. However, we did not find any highly expressed genes involved in the hormone pathway in long-spurred flowers. In contrast to previous hypotheses that anisotropic cell expansion leads to interspecific spur variation in Aquilegia, our results suggest that cell number changes and related genes are mainly responsible for spur length variations of A. rockii. Furthermore, the underlying mechanisms of similar floral traits in morphology may be quite different, enriching our understanding of the mechanisms of flower diversity in angiosperms.

Floral scent emitted by white and coloured morphs in orchids.

Polymorphism of floral signals, such as colour and odour, is widespread in flowering plants and often considered to be adaptive, reflecting various pollinator preferences for particular floral traits. Several authors have recently hypothesized that particular associations exist between floral colour and scent, which would result from shared biochemistry between these two floral traits. In this study, we compared the chemical composition of floral volatiles emitted by white- and purple-flowered morphs of three different orchid species, including two food-deceptive species (Orchis mascula and Orchis simia) and a food-rewarding species (Anacamptis coriophora fragrans). We found clear interspecific differences in floral odours. As expected from their pollination strategy, the two deceptive orchids showed high inter-individual variation of floral volatiles, whereas the food-rewarding A. c. fragrans showed low variation of floral scent. Floral volatiles did not differ overall between white- and coloured-flowered morphs in O. mascula and A. c. fragrans, while O. simia exhibited different volatile profiles between the two colour morphs. However, a detailed analysis restricted to benzenoid compounds (which are associated with the production of floral anthocyanin pigments) showed that white inflorescences emitted more volatiles of the shikimic pathway than coloured ones, both for O. mascula and O. simia. These results are consistent with the current hypothesis that shared biochemistry creates pleiotropic links between floral colour and scent. Whether intraspecific variation of floral signals actually affects pollinator attraction and influences the reproductive success of these orchids remains to be determined.

FERTILITY SELECTION ON A DISCRETE FLORAL POLYMORPHISM IN CLARKIA (ONAGRACEAE).

This study investigated fertility selection on a flower petal pigmentation polymorphism in Clarkia gracilis ssp. sonomensis. Natural populations are typically composed of nearly 100% spotted-petal plants, although rare populations contain a majority of unspotted plants. I compared fitness values for the two morphs using a simple fertility model to estimate selection for experimental arrays of plants placed into existing populations of different phenotypic frequencies. Both male and female reproductive success were estimated as well as the pattern of mating among phenotypes. Although the separate fitness components varied from no differences to a strong advantage for spotted plants, for every situation the selection calculations predicted an increase in the frequency of the spotted allele. Pollinator behavior and postpollination mechanisms may be responsible for the fitness differences. The apparent inability of the unspotted allele to spread though most natural populations is consistent with its selective disadvantage in this study.

STOCHASTIC LOSS OF STYLE MORPHS FROM POPULATIONS OF TRISTYLOUS LYTHRUM SALICARIA AND DECODON VERTICILLATUS (LYTHRACEAE).

Despite the theoretical importance of stochastic processes in evolution there have been few empirical studies of the interaction between genetic drift and selection on the maintenance of polymorphisms in plant populations. We used computer models to investigate the interaction between drift and frequency-dependent selection in affecting style morph frequencies in populations of tristylous species. Drift produces a distinct pattern of morph frequency variation involving: 1) the loss of the S morph and, to a lesser extent, the M morph; 2) no consistent bias in frequencies within populations; 3) a restricted pattern of variation involving a deficiency of one morph and equal excesses of the other two. Morph frequencies were surveyed in 137 populations of Lythrum salicaria from both its native range in Europe (N = 35) and recent adventive range in Ontario (N = 102), and 133 populations of Decodon verticillatus from four regions in eastern North America with different glacial histories to assess these theoretical predictions. There was a negative relationship between morph loss and population size in both species; the relationship was weaker in D. verticillatus than in L. salicaria. Morph loss was more frequent in the adventive than native range of L. salicaria, and in populations of D. verticillatus from glaciated northern regions compared with the unglaciated southern portion of its range. Simulations incorporating variation in life history, regeneration strategy and mating patterns revealed that the degree of morph loss was strongly influenced by year to year survival, clonal propagation, self-fertilization and departures from disassortative mating. Comparing the pattern of morph frequency variation between species supported these predictions. Morph loss was lower in self-incompatible L. salicaria (0% in Europe; 23% in Ontario), which reproduces through seed compared to self-compatible, clonal D. verticillatus (52%). A stochastic model provides the most parsimonious explanation for observed patterns of morph frequency variation in both species.

GENETIC DRIFT AND FOUNDER EFFECT IN NATIVE VERSUS INTRODUCED POPULATIONS OF AN INVADING PLANT, LYTHRUM SALICARIA (LYTHRACEAE).

There are few convincing examples of genetic drift at loci under selection in natural populations. The plant sexual polymorphism tristyly provides an opportunity to investigate genetic drift because stochastic processes interacting with frequency-dependent selection give rise to a diagnostic pattern of morph-frequency variation. A previous study of 102 Ontario populations of the introduced tristylous wetland herb Lythrum salicaria provided evidence for the role of stochastic processes during colonization. However, whether stochastic effects are greater in these recently introduced populations compared to native Eurasian populations remains unclear. The propensity of this species to invade disturbed habitats suggests that episodes of colonization and periods of small population size must also occur in the native range. A survey of 102 populations in southwestern France indicated reduced stochastic effects in native populations. Populations exhibited significantly lower morph loss than in Ontario (5% vs. 23%) and significantly higher values of morph evenness. The greater incidence of trimorphism in French populations was not associated with larger population sizes; populations were significantly smaller than those in Ontario (means: 266 vs. 487). Morph evenness was positively correlated with population size among French but not Ontario populations, providing further evidence of nonequilibrium conditions in introduced compared to native populations. The incidence of trimorphism was unexpectedly high in small native populations (N ≤ 25; 22 of 27 populations trimorphic). Computer simulations indicated that levels of gene flow on the order of m ≥ 0.05 can account for the maintenance of tristyly in small populations. The high connectivity of populations within the agricultural landscape typical of southwestern France may facilitate levels of gene flow sufficient to maintain trimorphism in small populations.